The authors would like to thank Mariko Yamatake and Masako Takashima for technical assistance. This work was supported in part by KAKENHI grant numbers 16H00759 and 17H04017 (SO).

1. Prepare Liposomes
<ol>
	<li>Disperse phospholipids (e.g., 10 mg in powder) in chloroform at a desired concentration (e.g., 10 mg/mL).</li>
	<li>Evaporate chloroform.
	<ol>
		<li>Place the phospholipid solution in a round-bottom flask and set it on a rotary evaporator (see Table of Materials) connected to a N2 gas cylinder. Rotate the flask under N2 flow at room temperature until a thin phospholipid film appears (after ~30 min).</li>
		<li>Place the open flask ...

The CBB method of lipid bilayer formation is based on the principle of a water-in-oil droplet lined by a monolayer20. Technically, the procedures for forming CBBs are easy, especially for patch-clamp researchers, who are proficient in manipulating glass micropipettes. The electrophysiological setup for the patch clamp is readily used in the CBB when two pipette manipulators with microinjectors are available. On the other hand, because the CBB is a successor of the conventional PLB, for which a lar...

For ion channels, the cell membrane is not simply a supporting material but a partner for generating the ion flux. Functionally, the membrane is an electrical insulator in which ion channels are embedded, and all cell membranes are imparted with a resting membrane potential. Conventionally, an arbitrary membrane potential was imposed from an external circuit by which electrical current through the channels was measured. This quantitative evaluation of the ion flux at different membrane potentials revealed the molecular properties of these channels, such as their ion-selective permeation and gating functions1,2. The membrane platform for functional studies of ion channels is either the cell membrane or the lipid bilayer membrane. Historically, single-channel electrical current recordings were first performed in lipid bilayers3,4, and the relevant techniques were developed for cell membranes, such as the patch-clamp method (Figure 1A)5,6. Since then, these two techniques have evolved separately for different purposes (Figure 1)7,8.
Membrane lipids and bilayer membranes are currently the focus of research for their roles in supporting the structure and function of channel proteins. Therefore, the ready availability of methods to vary the lipid composition in bilayers is in high demand. Lipid bilayer formation methods such as the planar lipid bilayer (PLB)8,9,10,11, water-in-oil droplet bilayer12, and droplet interface bilayer (DIB)13,14,15,16,17,18,19 techniques (Figure 1) are common choices, providing an opportunity for examining the channel function under varying lipid compositions20. Although the DIB is technically much easier to produce than the conventional PLB, the large size of the DIB has created a disincentive for patch-clampers to apply it for studying single-channel current recordings with usual-sized conductance (&#60;100 pS).
To circumvent the background noise, the bilayer area must be minimized. This issue recalls the repetitions of history in developing electrophysiological techniques for lipid bilayers (Figure 1). In the early days, a small-sized bilayer (1-30 &#181;m in diameter) was formed at the tip of a pipette (tip-dip method; Figure 1C)21,22,23, rather than using a free-standing bilayer (~100 &#181;m in diameter) on a hydrophobic septum in a chamber (Figure 1B). The tip-dip method allowed for electrical measurements with much lower background noise24. Our experiences with the PLB25,26, tip-dip22,23,27, and patch-clamp28,29,30,31 methods led us to a novel idea of forming lipid bilayers by using the principles of the water-in-oil bilayer. We have referred to this as the contact bubble bilayer (CBB) method20,32. In this method, rather than hanging the water droplets in an oil phase (Figure 1D), a water bubble is blown from a glass pipette (with tip diameter of approximately 30 &#181;m) into the oil phase (Figure 1E and 2), where the bubble is maintained by applying a steady pressure. A monolayer forms spontaneously at the water-oil interface at the surface of the bubble. Then, two bubbles are docked through the manipulation of two glass pipettes, and the bilayer is formed as the two monolayers approach each other, yielding an equilibrium bilayer area. The size of the bubble is controlled by the intra-bubble pressure (holding pressure), and likewise the bilayer size. An average diameter of 50 &#181;m is frequently used. Although the volume of the bubble is small (&#60;100 pL), it is connected to the larger volume of the pipette solution that is in the microliter range, constituting the bulk electrolyte phase.
There are many benefits to use the CBB method (Table 1). As a lipid bilayer formation technique, membranes of various lipid compositions can be produced, and asymmetric membranes are more readily formed32 than are those by the conventional folding method33. The bilayer can be mechanically manipulated, unlike the conventional PLB that can only be bent with a hydrostatic pressure difference34,35. By changing the holding pressure, the bubbles either expand or shrink, leading to increased or decreased membrane tension32. The bilayer is mechanically detachable into monolayers, similar to the freeze-fracture technique36,37 of membranes in morphological studies, but with the CBB, a maneuver allows for repeated detach and attach cycles32. The small volume of the electrolyte solution within the bubble allows efficient fusion of channel-reconstituted liposomes into the bilayer, and the probability of getting channel recordings is much higher than with the conventional PLB technique. The small bubble volume also allows rapid perfusion (within ~20 ms) once another injection pipette is inserted into either of the bubbles. Unlike the patch-clamp method, once broken, a CBB membrane is re-formed immediately and repeatedly, and pipettes can be used several times a day. By integrating benefits of the patch-clamp and PLB methods, the CBB provides a versatile platform to vary the physicochemical conditions of the membrane, allowing for unprecedented studies of channel-membrane interactions.
Before presenting a detailed protocol of the CBB formation process, the physicochemical background of the bilayer formation is presented first,which will be helpful for patch-clampers to resolve experimental difficulties relating to the membrane formation that are encountered.
CBB experiments impart lessons of surface chemistry science38. The CBB is similar to a soap bubble blown from a straw into the air, where likewise, a water bubble is blown into an organic solvent. One will notice that a water bubble is hardly inflated when membrane lipids are not included in either the water bubble or the organic solvent. In the absence of amphipathic lipids, the surface tension at a water-oil interface is high, and the intra-bubble pressure for blowing a bubble will be high. This is a realization of the Laplace equation (&#916;P = 2 &#947;/R, where &#916;P is the intra-bubble pressure, &#947; is the surface tension, and R is the bubble radius). When the concentration of lipids in either the organic phase or the electrolyte solution is high, the density of lipids in the monolayer increases, as dictated by the Gibbs adsorption isotherm (-d&#947; = &#915;i d&#181;i, where &#915;i is the surface excess of compound i, and &#181;i is the chemical potential of component i)39, leading to a lower surface tension and ease of bubble formation. In the CBB, the bilayer can be observed from a tangential angle (Figure 2), and the contact angle between the monolayer and bilayer is measurable. This angle represents an equilibrium between the surface tensions of the monolayer and bilayer (Young equation: &#947;bi = &#947;mo cos(&#952;), where &#947;bi is the bilayer tension, &#947;mo is the monolayer tension, and &#952; is the contact angle). The changes in the contact angle indicate changes in the bilayer tension, given that the monolayer tension is evaluated from changes in the contact angle as a function of the membrane potential (Young-Lippmann equation: &#947;mo = Cm V2/4 (cos(&#952;0) - cos(&#952;v)), where Cm is the membrane capacitance, V is the membrane potential, and &#952;0 and &#952;v are the contact angles at 0 and V mV, respectively)40,41,42. When two bubbles are close enough, they approach each other spontaneously. This is due to the van der Waals force, and we can visually observe this dynamic process in CBB formation.
A CBB system consists of distinct phases: namely, a bulk oil phase, water bubbles coated with a monolayer, and a contacting bilayer (Figure 3). These are reminiscent of the multiple phases observed in a PLB, such as a solvent-containing torus around the bilayer phase and a thin organic phase sandwiched by two monolayers43,44. In the CBB, the monolayer phase is continuous with the bilayer leaflet, and lipid molecules readily diffuse between the monolayer and the leaflet. The monolayer phase covers most of the bubble surface, constituting the major phase that serves as a lipid reservoir. Because the hydrophobic tail of lipids in the monolayer extends outward to the bulk oil phase, the bilayer interior or the hydrophobic core opens to the bulk oil phase. Thus, a hydrophobic substance injected into the oil phase close to the bilayer is able to readily access the bilayer interior. This is the membrane perfusion technique we had developed recently45, by which the lipid composition in the bilayer is changed rapidly (within a second) during single-channel current recordings. We found that the cholesterol content in the bilayer could be reversibly controlled by switching the cholesterol perfusion on and off45. In the event that the concentration of the relevant substance in the monolayer and bilayer differs, the concentration gradient of the relevant substance is immediately dissolved through diffusion, which is known as the Marangoni effect46,47. On the other hand, flip-flops across the monolayers are slow48,49,50.
Using the CBB method, the bilayer is formed under versatile physicochemical conditions, such as an electrolyte pH as low as 1 51, a salt (K+, Na+, etc.) concentration up to 3 M, a membrane potential as high as &#177;400 mV, and a system temperature of up to 60 &#176;C.
There are several options for formation of the CBB and incorporation of channel molecules therein. For formation of the monolayer at the water-oil interface, lipids are added either in an organic solvent (lipid-out method; Figure 4A, 4C) or in a bubble as liposomes (lipid-in method; Figure 4B, 4D). Notably, the lipid-in method allows for the formation of asymmetric membranes15,32. Channel molecules soluble in aqueous solution (e.g., channel-forming peptides) are directly added into the bubble (Figure 4A, B)52,53, whereas channel proteins are reconstituted into liposomes, which are then added into the bubble (Figure 4C, D). Herein, the formation of CBBs by the lipid-in method for either a channel peptide (polytheonamide B (pTB); Figure 4A) or a protein (KcsA potassium channel, Figure 4C) is shown.

<table border="0" cellpadding="0" cellspacing="0" style="width:607px;" width="605">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">Azolectin (L-&#945;-Phosphatidylcholine, Type IV-S)</td>
			<td style="width:85px;">Sigma-Aldrich</td>
			<td style="width:124px;">P3644</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:219px;">A/D Converter</td>
			<td style="width:85px;">Molecular Divices</td>
			<td style="width:124px;">Digidata1550A</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">Ag/AgCl electrode</td>
			<td style="width:85px;">Warner Instruments</td>
			<td style="width:124px;">64-1317</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Bath Sonicator</td>
			<td style="width:85px;">Branson</td>
			<td style="width:124px;">M1800H-J</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">Camera</td>
			<td style="width:85px;">Hamamatsu Photonics</td>
			<td style="width:124px;">C11440-10C</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:219px;">Glass Capillary</td>
			<td style="width:85px;">Harvard Apparatus</td>
			<td style="width:124px;">30-0062</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Hepes</td>
			<td style="width:85px;">Dojindo</td>
			<td style="width:124px;">342-01375</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:219px;">Hole Slideglass</td>
			<td style="width:85px;">Matsunami Glass</td>
			<td style="width:124px;">S339929</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Inverted Microscope</td>
			<td style="width:85px;">Olympus</td>
			<td style="width:124px;">IX73</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Isolation Table</td>
			<td style="width:85px;">Herz</td>
			<td style="width:124px;">TDI-86LA(Y)2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Micro Injenctor</td>
			<td style="width:85px;">Narishige</td>
			<td style="width:124px;">IM-11-2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Micro Manipulator</td>
			<td style="width:85px;">Narishige</td>
			<td style="width:124px;">EMM</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Microforge</td>
			<td style="width:85px;">Narishige</td>
			<td style="width:124px;">MF-830</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Micropipette holder</td>
			<td style="width:85px;"></td>
			<td style="width:124px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">n-Hexadecane</td>
			<td style="width:85px;">Nacalai</td>
			<td style="width:124px;">07819-32</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Patch-Clamp Amplifier</td>
			<td style="width:85px;">HEKA</td>
			<td style="width:124px;">EPC800</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">Pipette Puller</td>
			<td style="width:85px;">Sutter Instrument Co.</td>
			<td style="width:124px;">P-87</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine)</td>
			<td style="width:85px;">Avanti Polar Lipids</td>
			<td style="width:124px;">850457</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:219px;">POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine 
			)</td>
			<td style="width:85px;">Avanti Polar Lipids</td>
			<td style="width:124px;">850757</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) )</td>
			<td style="width:85px;">Avanti Polar Lipids</td>
			<td style="width:124px;">840457</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Potassium Chloride</td>
			<td style="width:85px;">Nacalai</td>
			<td style="width:124px;">28514-75</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Rotary Evapolator</td>
			<td style="width:85px;">Iwaki</td>
			<td style="width:124px;">REN-1000</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Succinic Acid</td>
			<td style="width:85px;">Nacalai</td>
			<td style="width:124px;">32402-05</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Vacuum Pump</td>
			<td style="width:85px;">Buchi</td>
			<td style="width:124px;">V-100</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

lipid bilayer experiments with contact bubble bilayers for patch-clampers

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 &#181;m in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.

A typical CBB had a diameter of 50 &#181;m (Figure 5, 6) and the specific membrane capacitance in hexadecane was 0.65 &#181;F/cm2. The bubble size was arbitrarily controlled by the intra-bubble pressure. When small bubbles are necessary for low-noise recordings, the tip diameter should be correspondingly small. For example, for a bubble size of 50 &#181;m in diameter, the tip diameter should be 30 &#181;m.
<p class="jove_conten...

Watch this Scientific Journal Video about Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers at JoVE.com

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Here, we present a protocol for the formation of lipid bilayers using a contact bubble bilayer method. A water bubble is blown into an organic solvent, whereby a monolayer is formed at the water-oil interface. Two pipettes are manipulated to dock the bubbles to form a bilayer.

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...

The Contact Bubble Bilayer method is an alternative technique for conventional Lipid Bilayers. and this method allows more versatile vibration through the membrane. The CBB method integrates the benefits of Planar Lipid Bilayer and patch clamp method.As a lipid bilayer technique a CBB with an arbitrary lipid composition can be formed. We have developed a CBB for single channel current recordings with high signal to noise ratio. This method allows varieties of membrane vibrations and offers a vast type platform to the channel membrane interface.Everybody has experienced blowing a soap bubble. Similar maneuvers are taken for CBB formation by manually fine-tuning the applied pressure rather than blowing by mouth. Practice and enjoy the CBB.To being this procedure disperse phospholipids in chloroform at a desired concentration in a round bottom flask. Place the phospholipid solution on a rotary evaporator connected to a nitrogen gas cylinder. Rotate the flask under nitrogen flow at room temperature until a thin phospholipid film appears.Next place the open flask into a desiccator that is connected to a vacuum pump. Using the vacuum pump aspirate the the inside of the desiccator for several hours to remove the chloroform thoroughly. Subsequently add an appropriate volume of electrolyte solution to the flask and suspend the phospholipid to obtain a two milligrmas per milliliter phospholipid suspension.Sonciate the suspension for 20 to 30 seconds using a bath sonicator to obtain a multilayered vesicle suspension. For the preparation of proteoliposomes that contain ion channel proteins, add a protein solution to the multilayered vesicle suspension. And sonicate for several seconds using the bath sonicator.To prepare large bore glass pipettes, place a glass capillary in a pipette puller and fabricate the micropipette with a fine tapered tip through two step pulling. Then set the micropipette on a micro forge and contact the tip of the micropipette to a platinum filament at the tapered portion with a diameter of 30 to 50 micrometers. Heat the filament briefly and immediately turn it off.Using distilled water and ethanol clean the surface of the glass slide with a shallow well. Next apply the siliconizing reagent on to the glass slide and let the reagent dry completely in the air. Then place the glass slide on the stage of an inverted microscope.Add 100 microliters of hexadecane into the shallow well of the siliconized glass slide. Using a tuberculin syringe fill the electrolyte solution up to half the length of the micropipette. Next set the micropipette onto the micropipette holder with the pressure port, allowing the silver/silver chloride wire electrode to soak in the pipette electrolyte solution.Connect one of the micropipette holders to the head stage of a patch clamp amplifier and the other one to the electrical ground. Subsequently connect a microinjector to the pressure port of the micropipette holder. Adjust the micropipette to an appropriate position above the stage of an inverted microscope using the micromanipulator.To adjust the electrode offset potential, place one microliter of the same electrolyte solution used to fill the micropipette on the flat surface around the shallow well of the glass slide to create an electrolyte dome. Soak the tip of both micropipettes in the electrolyte dome. Afterward adjust the electrode offset potential of the patch clamp amplifier.To draw the liposome solution from the tip, add one microliter of liposome solution on the flat surface around the shallow well of the slide glass. Next place the tip of the micropipette in the liposome containing dome. Aspirate the liposome containing solution using the microinjector.Repeat the procedure for the other mircopipette for aspirating channel reconstituted liposomes. Now place the of the micropipette in the hexadecane in the shallow well. Blow a water bubble slowly by increasing the pressure until the bubble reaches the desired size and maintain the same pressure thereafter.Discard the bubble by passing the tip through the oil-air interface if it is hard to keep the size of the bubble stable. Repeat the procedures until two stable bubbles are formed. Establish a contact between the two bubbles.Fine-tune the pressure to maintain the bubble size, because the size may gradually change even at the constant intra-bubble pressure. Set the membrane potential to the appropriate value using the patch claim amplifier and wait for the channel current to emerge. For stable lipid bilayer formation it is essential to clean the blotters.Glass capillary or pipette preparation should be washed thoroughly and avoid contamination by the detergent molecules. Once the CBB had formed the channel molecules either in aqueous solution or in the liposome were spontaneously inserted into the bilayer within the span of a few to dozens of minutes. Insertions of the channels was confirmed by the step wise increase of current amplitude under the applied membrane potential.A bilayer formed by the CBB method can be disintegrated into two monolayers. The pTB channel current emerged immediately after attaching the two monolayers. And the current became larger as the are of contact increased the amplitude of the current was synchronized with the detach attach manipulation of the CBB.If the number blow outs of bubbles increases the lipid concentration in the aqueous solution decreases and the monolayer is not formed. Then try to relieve pressure on liposome section. Once the CBB formation and channel reconstitution are established you can try to vary compositions of aqueous solution and membrane via profusion or injecting a hydrophilic or hydrophobic substances.The CBB method has opened versatile ways to explore channel-membrane interplays.

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9.4K Views.  University of Fukui Faculty of Medical Sciences. The Contact Bubble Bilayer method is an alternative technique for conventional Lipid Bilayers. and this method allows more versatile vibration through the membrane. The CBB method integrates the benefits of Planar Lipid Bilayer and patch clamp method.As a lipid bilayer technique a CBB with an arbitrary lipid composition can be formed. We have developed a CBB for single channel current recordings with high signal to noise ratio. This method allows varieties of membrane vibrations and of...